Isolation and partial characterization of the Asp-B10, Tyr-B25-des-(B26-B30)-proinsulin analog from inclusion bodies in Escherichia coli

Summary A proinsulin analog constructed by site-directed mutagenesis was expressed as a fusion protein that formed inclusion bodies inside the cells. It was purified from the isolated inclusion bodies and proinsulin was obtained by trifluoro-acetic acid, dimethyl sulfoxide and hydrochloric acid cleavage. The released proinsulin analog was confirmed by its molecular weight as determined by SDS-PAGE.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Application of life cycle assessment to landfilling

A case study of a life-cycle assessment (LCA) is performed concerning the treatment of household solid wastes in a landfill. The stages considered in this LCA study are: goal and scope definition, inventory analysis and impact assessment. The data of the inventory include the consumption of raw materials and energy through the transport of wastes and the management of landfill, and the corresponding emissions to the environment. Abiotic resource depletion, global warming, acidification, eutrophication and human toxicological impacts have been considered as impact categories for the impact assessment phase of the LCA. A comparison of the environmental impact of the landfilling with and without energy recovery is carried out. Members of the Spanish Association for LCA Development (APRODACV)     

A proposed architecture for the central domain of the bacterial enhancer-binding proteins based on secondary structure prediction and fold recognition

The expression of genes transcribed by the RNA polymerase with the alternative sigma factor <r54 (Ecr54) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter. These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis. The best characterized proteins of this family of regulators are NtrC and Nif A, which activate genes required for ammonia assimilation and nitrogen fixation, respectively. In a recent IRBM course (“Frontiers of protein structure prediction,” IRBM, Pomezia, Italy, 1995; see web site http://www.mrc-cpe.cam.uk/ irbm-course95/), one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold. This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology. In this work, we present new data that support the previous conclusion. The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alfi topology. The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence. Finally, and most importantly, three of five reported residue alterations that impair the Central domain ATPase activity of the Eo-54 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotide-binding in the mononucleotide-binding proteins. Furthermore, the known residue substitutions that alter the function of the Ecr54 activators, leaving intact the Central domain ATPase activity, are mapped on a region proposed to play an equivalent role as the effector region of the GTPase superfamily.     

Korean and Caucasian overweight premenopausal women have different relationship of body mass index to percent body fat with age.

The aim of the study was to investigate in premenopausal women whether the relationship between percentage body fat (PBF) and body mass index (BMI; in kg/m2) differs between Korean Asians (Ko-As) living in Seoul, South Korea, and Caucasians (Ca) living in New York City. Healthy premenopausal women (50 Ko-As; 38 Ca), ages 22-50 yr, were studied. Weight, height, and PBF by dual-energy X-ray absorptiometry were measured. Total body dual-energy X-ray absorptiometry data were collected using GE-Lunar systems (Prodigy-Korea and DPXL-New York), and all scan analyses were performed by one technician in New York. Similar soft tissue phantoms were used for daily instrument calibrations at both sites. The relationship between PBF and BMI was assessed by multiple regression analysis with race, age, reciprocal of BMI (1/BMI), and a race-by-age interaction as the final independent variables. Race (P = 0.003) and 1/BMI (P < 0.001) were significantly related to PBF in this model. A significant race-by-age interaction (P = 0.039) indicated that the slope of the lines for PBF vs. age differed between Ko-As and Ca. This study demonstrates in a Ko-As sample that the BMI-fat relationship differs significantly from that in a comparable group of Caucasian women. Investigators who use BMI as an index of fatness should be aware of the well documented differences in the relationship of BMI and fatness across race/ethnic groups.     

Genetic markers validate using the natural phenotypic characteristics of shed feathers to identify individual northern goshawks Accipiter gentilis

The recognition of individual animals is essential for many types of ecological research, as it enables estimates of demographic parameters such as population size, survival and reproductive rates. A popular method of visually identifying individuals uses natural variations in spot, stripe or scar markings. Although several studies have assessed the accuracy of these methods in mammals, crustaceans and fish, there have been few attempts to determine whether phenotypic characteristics are accurate when used for birds. Furthermore, even less is known about whether shed or moulted body parts can be reliably used to visually identify individuals. Here we assessed the accuracy of using phenotypic characteristics to identify avian individuals using a double‐marking experiment, whereby nine microsatellite genetic markers and natural markings on shed feathers were used to independently identify northern goshawks Accipiter gentilis. Phenotypic and genetic identification of individuals was consistent in 94.4% (51/54) comparisons. Our results suggest that the phenotypic characteristics of shed feathers can be reliably used as a non‐invasive and relatively inexpensive technique to monitor populations of an elusive species, the northern goshawk, without having to physically re‐capture or re‐sight individuals. We posit that using natural markings on shed feathers will also be a reliable method of identifying individuals in avian species with similar phenotypic characteristics, such as other Accipiter species.     

A novel archaeal DNA repair factor that acts with the UvrABC system to repair mitomycin C‐induced DNA damage in a PCNA‐dependent manner

The sliding clamp proliferating cell nuclear antigen (PCNA) plays a vital role in a number of DNA repair pathways in eukaryotes and archaea by acting as a stable platform onto which other essential protein factors assemble. Many of these proteins interact with PCNA via a short peptide sequence known as a PIP (PCNA interacting protein) motif. Here we describe the identification and functional analysis of a novel PCNA interacting protein NreA that is conserved in the archaea and that has a PIP motif at its C‐terminus. Using the genetically tractable euryarchaeon Haloferax volcanii as a model system, we show that the NreA protein is not required for cell viability but that loss of NreA (or replacement of the wild‐type protein with a truncated version lacking the C‐terminal PIP motif) results in an increased sensitivity to the DNA damaging agent mitomycin C (MMC) that correlates with delayed repair of MMC‐induced chromosomal DNA damage monitored by pulsed‐field gel electrophoresis. Genetic epistasis analysis in Hfx. volcanii suggests that NreA works together with the UvrABC proteins in repairing DNA damage resulting from exposure to MMC. The wide distribution of NreA family members implies an important role for the protein in DNA damage repair in all archaeal lineages.